Isoprenoid analog compounds and methods of making and use thereof

ABSTRACT

The invention provides compounds of formula I:  
                 
 
     wherein X, R 1 , R 2 , and n have any of the values defined in the specification, and their pharmaceutically acceptable salts. The compounds are useful, for example, for blocking prenylation transferase enzymes, for probing or diagnosing protein prenylation processes, and for treating cancer in a mammal. The invention also provides pharmaceutical compositions, processes for preparing compounds of formula I, and intermediates useful for the synthesis of compounds of formula I.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional under 37 C.F.R. 1.53(b) of U.S.patent application Ser. No. 10/116,737 filed Apr. 3, 2002, which claimedpriority under 35 U.S.C. 119(e) to U.S. Provisional Patent ApplicationSerial No. 60/281,170, filed Apr. 3, 2001, which applications areincorporated by reference and made a part hereof.

BACKGROUND OF THE INVENTION

[0002] Prenylation of proteins is a common form of post-translationalprocessing found in many biological systems. For example, at least 300human proteins are prenylated and many of these proteins play criticalroles in essential signal transduction pathways. Mevalonic acidderivatives, known collectively as isoprenoids, are known to be centralto mammalian metabolism, but the smaller intermediates in this pathwaywere viewed primarily as precursors to larger compounds such as steroidsand dolichols. Not until 1989 was the presence of prenylated cysteinesdemonstrated in mammalian cells. The number of prenylated proteinscontinues to grow, with sequences for at least 300 prenylated proteinsnow recognized in humans corresponding to about 1% of the total cellularprotein by mass. These include all of the known monomeric and trimeric Gproteins, proteins which play central roles in a variety of signaltransduction processes that regulate cell growth. Therefore, inhibitionor modification of protein prenylation processes is a promising area forthe development of new therapeutic agents and treatment methods, forexample, anticancer agents and cancer treatments. Additionally,diagnosis or treatment regimes directed toward inhibition ormodification of protein prenylation processes can be improved andrefined with the help of enhanced means and methods of detecting changesin vivo or in vitro in the prenylation process.

[0003] Goody reported the preparation of detectable terpenoid analogcompounds as substrates for GGPTase II in vitro. (Angew. Chem., Int. Ed.Eng., 1999, 38, 509-512; See also J. Davisson, et al. J. Org. Chem.1986, 51, 4768-4779.) However, potential biologic and chemicalinstability considerations of these compounds may limit their utility,for example, in in vivo experiments where intracellular esterases cancleave the anthranilic ester linkage to the component parts therebyliberating a fluorescent anthranilic acid.

[0004] Other publications of interest are listed in the referencessection below.

[0005] U.S. Pat. Nos. 5,998,204 and 6,197,928 are of general interestand relate to fluorescent protein sensors for detection of analytes, forexample, localization sequences for prenylation or for insertion into aplasma membrane ([CaaX] CAAX (SEQ ID NO:51).

[0006] Thus, there is a continuing need for compounds which can inhibitthe prenylation process, and have improved stability, improved spectraldetectability, or both.

SUMMARY OF THE INVENTION

[0007] The present invention provides analog compounds of keyintermediates of isoprenoid biosynthesis and metabolism. These analogscan be prepared through chemical synthesis and can function as alternatesubstrates for enzymes involved in post-translation processing in eitherin vitro or in vivo. The compounds of the invention can be potentprenylation process inhibitor compounds which can also have improvedstability, improved spectral detectability, or both. The compounds ofthe invention are also useful as probes for studying the prenylationprocess and related processes.

[0008] The invention provides a compound of formula I:

[0009] wherein:

[0010] X is independently —NR_(a)—, O, or S;

[0011] R₁ is a detectable group;

[0012] R₂ is independently

[0013] OH,

[0014] (C₁-C₁₀)alkanoyloxy,

[0015] —O—P(═O)(—OR_(a))₂,

[0016] —O—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂,

[0017] —CH₂—O—P(═O)(—OR_(a))₂,

[0018] —CH₂—O—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂,

[0019] —CH₂—P(═O)(—OR_(a))₂,

[0020] —CH{—P(═O)(—OR_(a))₂}₂,

[0021] —CH₂—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂,

[0022] —CH═CH {—P(═O)(—OR_(a))₂}, or

[0023] —CH═C {—P(═O)(—OR_(a))₂}₂;

[0024] each R_(a) is independently hydrogen, (C₁-C₁₀)alkyl,(C₁-C₁₀)alkanoyl, (C₁-C₁₀)alkanoyloxy, (C₁-C₁₀)alkoxycarbonyl, or—CH₂—O—(C₁-C₁₀)alkanoyl;

[0025] n is independently 1, 2, or 3;

[0026] or a pharmaceutically acceptable salt thereof.

[0027] The invention also provides a pharmaceutical compositioncomprising a compound of formula I and a pharmaceutically acceptablediluent or carrier.

[0028] The invention also provides a method of treating cancer,comprising administering to a mammal afflicted with cancer, an amount ofa compound of the invention effective to treat the cancer.

[0029] The invention also provides a method of inhibiting a prenylationtransferase or synthase enzyme comprising contacting the enzyme in vivoor in vitro with an effective amount of a compound of the invention.

[0030] The invention also provides a method of accessing the metabolicstatus of an enzyme, such as a prenylation transferase enzyme,comprising:

[0031] contacting the enzyme with an effective amount of a mixture of afarnesol analog compound of the invention and a geraniol orgeranylgeraniol analog compound of the invention, and as describedherein; and

[0032] measuring the relative ratio, or levels, of farnesylation togeranylgeranylation of the farnesol and geraniol or geranylgeraniolanalog compounds accomplished by the enzyme, and wherein the ratiocorrelates with the metabolic status of the enzyme.

[0033] The invention also provides a compound of the invention for usein medical therapy or diagnosis, for example, treating cancer.

[0034] The invention also provides for the use of a compound of theinvention for the manufacture of a medicament useful for the treatmentof cancer.

[0035] The invention also provides for the use of a compound of theinvention for the manufacture of a medicament useful for inhibitingprenylation transferase or synthase enzymes in a mammal.

[0036] The invention also provides processes and intermediates disclosedherein that are useful for preparing compounds of the invention. Somecompounds of the formula I are useful as intermediates in preparingother compounds of formula I.

BRIEF DESCRIPTION OF DRAWINGS

[0037]FIG. 1 schematically illustrates isoprenoid biosynthesis andprotein prenylation routes in embodiments of the present invention.

[0038]FIG. 2 schematically illustrates normal RAS farnesylation inembodiments of the present invention.

[0039]FIG. 3 schematically illustrates an FTPase mediated reaction ofRas with a vinyl phosphonate in embodiments of the present invention.

DETAILED DESCRIPTION

[0040] The following definitions are used, unless otherwise described:halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote bothstraight and branched groups; but reference to an individual radicalsuch as “propyl” embraces only the straight chain radical, a branchedchain isomer such as “isopropyl” being specifically referred to. Whenalkyl can be partially unsaturated, the alkyl chain may comprise one ormore (e.g. 1, 2, 3, or 4) double or triple bonds in the chain.

[0041] “Aryl” denotes a phenyl radical or an ortho-fused bicycliccarbocyclic radical having about nine to ten ring atoms in which atleast one ring is aromatic.

[0042] “Het” is a four-(4), five-(5), six-(6), or seven-(7) memberedsaturated or unsaturated heterocyclic ring having 1, 2, 3, or 4heteroatoms selected from the group consisting of oxy, thio, sulfinyl,sulfonyl, and nitrogen, which ring is optionally fused to a benzenering, or any cyclic heterocycle group which may be monocyclic ormulti-cyclic. Het includes “heteroaryl,” which encompasses a radicalattached via a ring carbon of a monocyclic aromatic ring containing fiveor six ring atoms consisting of carbon and 1, 2, 3, or 4 heteroatomseach selected from the group consisting of non-peroxide oxy, thio, andN(X) wherein X is absent or is H, O, C₁₋₄alkyl, phenyl or benzyl, aswell as a radical of an ortho-fused bicyclic heterocycle of about eightto ten ring atoms derived therefrom, particularly a benz-derivative orone derived by fusing a propylene, trimethylene, or tetramethylenediradical thereto.

[0043] The term “heterocycle” refers to a monovalent saturated orpartially unsaturated cyclic non-aromatic group which contains at leastone heteroatom, preferably 1 to 4 heteroatoms, selected from nitrogen(NR_(x), wherein R_(x) is hydrogen, alkyl, or a direct bond at the pointof attachment of the heterocycle group), sulfur, phosphorus, and oxygenwithin at least one cyclic ring and which may be monocyclic ormulti-cyclic. Such heterocycle groups preferably contain from 3 to 10atoms. The point of attachment of the heterocycle group may be a carbonor nitrogen atom. This term also includes heterocycle groups fused to anaryl or heteroaryl group, provided the point of attachment is on anon-aromatic heteroatom-containing ring. Representative heterocyclegroups include, by way of example, pyrrolidinyl, piperidinyl,piperazinyl, imidazolidinyl, morpholinyl, indolin-3-yl, 2-imidazolinyl,1,2,3,4-tetrahydroisoquinolin-2-yl, quinuclidinyl and the like.

[0044] The terms “include”, “for example”, “such as”, and the like areused illustratively and are not intended to limit the present invention.

[0045] The indefinite articles “a” and “an” mean “at least one” or “oneor more” when used in this application, including the claims, unlessspecifically indicated otherwise.

[0046] “Optional” or “optionally” mean that the subsequently describedevent or condition may but need not occur, and that the descriptionincludes instances where the event or condition occurs and instances inwhich it does not. For example, “optionally substituted” means that thenamed substituent may be present but need not be present, and thedescription includes situations where the named substituent is includedand situations where the named substituent is not included.

[0047] The compounds of the present invention are generally namedaccording to the IUPAC or CAS nomenclature system. Abbreviations whichare well known to one of ordinary skill in the art may be used (e.g.“Ph” for phenyl, “Me” for methyl, “Et” for ethyl, “h” for hour or hoursand “rt” for room temperature).

[0048] Specific and preferred values listed below for radicals,substituents, and ranges, are for illustration only; they do not excludeother defined values or other values within defined ranges for theradicals and substituents. The compounds of the invention includecompounds of formula I having any combination of the values, specificvalues, more specific values, and preferred values described herein.

[0049] The term “detectable group” refers to any known fluorophoresubstituent, for example, fluorescent groups including, but not limitedto, anthranilic acid compounds, aminonaphthalenesulfonic acid compounds,coumarin compounds, and like groups or compounds and as illustratedherein.

[0050] The term “treatment” refers to any treatment of a pathologiccondition in a mammal, particularly a human, and includes:

[0051] (i) preventing the pathologic condition from occurring in asubject which may be predisposed to the condition but has not yet beendiagnosed with the condition and, accordingly, the treatment constitutesprophylactic treatment for the disease condition;

[0052] (ii) inhibiting the pathologic condition, i.e., arresting itsdevelopment;

[0053] (iii) relieving the pathologic condition, i.e., causingregression of the pathologic condition; or

[0054] (iv) relieving the conditions mediated by the pathologiccondition.

[0055] The term “therapeutically effective amount” refers to that amountof a compound of the invention which is sufficient to effect treatment,as defined above, when administered to a mammal in need of suchtreatment. The therapeutically effective amount will vary depending uponthe subject and disease condition being treated, the weight and age ofthe subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art.

[0056] The term “pharmaceutically acceptable salts” includes, but is notlimited to, salts well known to those skilled in the art, for example,mono-salts (e.g. alkali metal and ammonium salts) and poly salts (e.g.di- or tri-salts,) of the compounds of the invention. Pharmaceuticallyacceptable salts of compounds of formula I are where, for example, anexchangeable group, such as hydrogen in —OH, —NH—, or —P(═O)(OH)—, isreplaced with a pharmaceutically acceptable cation (e.g. a sodium,potassium, or ammonium ion) and can be conveniently be prepared from acorresponding compound of formula I by, for example, reaction with asuitable base. In cases where compounds are sufficiently basic or acidicto form stable nontoxic acid or base salts, administration of thecompounds as salts may be appropriate. Examples of pharmaceuticallyacceptable salts are organic acid addition salts formed with acids whichform a physiological acceptable anion, for example, tosylate,methanesulfonate, acetate, citrate, malonate, tartarate, succinate,benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitableinorganic salts may also be formed, including hydrochloride, sulfate,nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptablesalts may be obtained using standard procedures well known in the art,for example, by reacting a sufficiently basic compound such as an aminewith a suitable acid affording a physiologically acceptable anion.Alkali metal (for example, sodium, potassium or lithium) or alkalineearth metal (for example, calcium) salts of carboxylic acids can also bemade.

[0057] It will be appreciated by those skilled in the art that compoundsof the invention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine transferase inhibitory activityusing the standard tests described herein, or using other similar testswhich are well known in the art. In particular, it is understood thatcompounds of formula I, such as R₁, R₂, or substituents thereon, canexist in the corresponding tautomeric “enol” form, and that suchtautomers are included as compounds of the invention.

[0058] Specific and preferred values listed below for radicals,substituents and ranges, are for illustration only; they do not excludeother defined values or other values within defined ranges for theradicals and substituents.

[0059] Specifically, (C₁-C₁₀)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl,octyl, nonyl, or decyl; (C₁-C₁₀)alkoxy can be methoxy, ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy,hexyloxy, heptyloxy, octyloxy, nonyloxy or decyloxy; (C₂-C₆)alkenyl canbe vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl,1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 4-hexenyl, or 5-hexenyl; (C₁-C₁₀)alkanoyl can be acetyl,propanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl,or decanoyl; (C₁-C₁₀)alkoxycarbonyl can be methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, hexyloxycarbonyl, heptyloxycarbonyl, octyloxycarbonyl,nonyloxycarbonyl or decyloxycarbonyl; (C₁-C₁₀)alkanoyloxy can beformyloxy, acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy,pentanoyloxy, hexanoyloxy, heptanoyloxy, octanoyloxy, nonanoyloxy, ordecanoyloxy; aryl can be phenyl, indenyl, naphthyl, or anthracenyl;heterocycle can benztriazolyl, triazinyl, oxazoyl, isoxazolyl,oxazolidinoyl, isoxazolidinoyl, thiazolyl, isothiazoyl, pyrazolyl,imidazolyl, pyrrolyl, pyrazinyl, pyridinyl, morpholinyl, quinolinyl,isoquinolinyl, indolyl, pyrimidinyl, piperidinyl, pyrrolidinyl,morpholinyl, thiomorpholinyl, or piperazinyl; and heteroaryl can be, forexample, furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl,thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl,1-methyl-1H-tetrazol-5-yl, pyridyl, (or its N-oxide), thienyl,pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) orquinolyl (or its N-oxide).

[0060] Specific and preferred values listed below for radicals,substituents, and ranges, are for illustration only; they do not excludeother defined values or other values within defined ranges for theradicals and substituents.

[0061] Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl.

[0062] Specifically, (C₁-C₆)alkoxy can be methoxy, ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, orhexoxy.

[0063] A specific compound of formula I is the formula II:

[0064] where R₁ is a 2-methoxycarboxy phenyl detectable group, R₂ isdiphosphate ester, n=1 to 3, and X is NH; or a pharmaceuticallyacceptable salt thereof.

[0065] Another specific compound of formula I is the formula III:

[0066] where n=0 to 3, and R is (C₁-C₁₀)alkyl, for example, methyl; or apharmaceutically acceptable salt thereof.

[0067] A specific value for R₁ is a detectable group, such as a knownfluorophore substituent.

[0068] A specific value for R₁ is an aryl group, such as phenyl,naphthyl, or anthracenyl, which aryl group is optionally substitutedwith one or more substituents independently selected from —COOR_(b),—S(O)_(n)NR_(b)R_(b), halo, cyano, nitro, aryl, heterocycle,(C₂-C₆)alkenyl, —C(═O)NR_(b)R_(b), —OC(═O)NR_(b)R_(b), —NR_(b)R_(b), or—S(O)_(n)R_(b), where each R_(b) is independently hydrogen,(C₁-C₁₀)alkyl, or (C₁-C₁₀)alkanoyl.

[0069] Another specific value for R₁ is Het, for example a heterocycleor heteroaryl, such as anthranil or quinoline, which Het is optionallysubstituted with one or more substituents independently selected from—COOR_(b), —S(O)_(n)NR_(b)R_(b), halo, cyano, nitro, aryl, heterocycle,(C₂-C₆)alkenyl, —C(═O)NR_(b)R_(b), —OC(═O)NR_(b)R_(b), —NR_(b)R_(b), or—S(O)_(n)R_(b), where each R_(b) is independently hydrogen,(C₁-C₁₀)alkyl, or (C₁-C₁₀)alkanoyl.

[0070] Another specific value for R₁ is substituted phenyl.

[0071] Another specific value for R₁ is phenyl substituted with a—COOR_(b).

[0072] Another specific value for R₁ is 2-methoxycarboxy phenyl.

[0073] Another specific value for R₁ is substituted naphthyl.

[0074] Another specific value for R₁ is naphthyl substituted with a—S(O)_(n)NR_(b)R_(b).

[0075] Another specific value for R₁ is naphthyl substituted at the5-position with a —S(O)_(n)NR_(b)R_(b) substituent.

[0076] Another specific value for R₁ is 5-N,N′-dimethylaminosulfonylnaphthy-1-yl.

[0077] A specific value for R₂ is OH.

[0078] Another specific value for is (C₁-C₁₀)alkanoyloxy.

[0079] Another specific value for R₂ is —O—P(═O)(—OR_(a))₂.

[0080] Another specific value for R₂ is—O—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂.

[0081] Another specific value for R₂ is —CH₂—O—P(═O)(—OR_(a))₂.

[0082] Another specific value for R₂ is—CH₂—O—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂.

[0083] Another specific value for R₂ is —CH₂—P(═O)(—OR_(a))₂.

[0084] Another specific value for R₂ is —CH{—P(═O)(—OR_(a))₂}₂.

[0085] Another specific value for R₂ is—CH₂—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂.

[0086] Another specific value for R₂ is —CH═CH{—P(═O)(—OR_(a))₂}.

[0087] Another specific value for R₂ is —CH═C{—P(═O)(—OR_(a))₂}₂.

[0088] A specific value for R_(a) is hydrogen.

[0089] A specific value for R_(a) is —C(═O)—CH₃.

[0090] Another specific value for R_(a) is —CH₃.

[0091] Another specific value for R_(a) is —CH₂—O—(C₁-C₆)alkanoyl.

[0092] A specific value for R_(b) is hydrogen.

[0093] Another specific value for R_(b) is —CH₃.

[0094] A specific value for n is 1.

[0095] Another specific value for n is 2.

[0096] Another specific value for n is 3.

[0097] A specific value for X is —NR_(a)—.

[0098] A specific value for X is —NH—.

[0099] A specific value for X is —N(CH₃)—.

[0100] A specific value for X is —O—.

[0101] A specific value for X is —S—.

[0102] A specific value for a protein conjugate of the present inventionis a protein linked to a fluorescent fragment of a compound of theinvention, for example, the Ras adduct of the compound of formula I,such as compounds of the formula II or III.

[0103] Processes and novel intermediates useful for preparing compoundsof formula I are provided as further embodiments of the invention andare illustrated by the following procedures in which the meanings of thegeneric radicals are as given above unless otherwise qualified.Compounds of formula I and preceding intermediates wherein R₁, R₂, and nhave any of the values, specific values, or preferred values definedherein, can be prepared in accordance with the preparative schemesdescribed below.

[0104] The invention provides a pharmaceutical composition, comprisingan effective amount of a compound of formula I as described hereinabove;or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier. The present compositions are preferably presented ina form suitable for absorption by the gastro-intestinal tract.

[0105] The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to a selected route ofadministration, i.e., by oral, parenteral, intravenous, intramuscular,topical, or subcutaneous routes. Thus, the present compounds may besystemically administered, e.g., orally, in combination with apharmaceutically acceptable vehicle such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the active compound may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2 toabout 60% of the weight of a given unit dosage form. The amount ofactive compound in such therapeutically useful compositions is such thatan effective dosage level will be obtained.

[0106] The tablets, troches, pills, capsules, and the like may alsocontain the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theactive compound may be incorporated into sustained-release preparationsand devices.

[0107] The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

[0108] The pharmaceutical dosage forms suitable for injection orinfusion can include sterile aqueous solutions or dispersions or sterilepowders comprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

[0109] Sterile injectable solutions are prepared by incorporating theactive compound in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

[0110] For topical administration, the present compounds may be appliedin pure form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

[0111] Useful solid carriers include finely divided solids such as talc,clay, microcrystalline cellulose, silica, alumina and the like. Usefulliquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the present compounds can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

[0112] Thickeners such as synthetic polymers, fatty acids, fatty acidsalts and esters, fatty alcohols, modified celluloses or modifiedmineral materials can also be employed with liquid carriers to formspreadable pastes, gels, ointments, soaps, and the like, for applicationdirectly to the skin of the user. Examples of useful dermatologicalcompositions which can be used to deliver the compounds of the inventionto the skin are disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392),Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157)and Wortzman (U.S. Pat. No. 4,820,508).

[0113] The present compositions may also be prepared in suitable formsfor absorption through the mucous membranes of the nose and throat orbronchial tissues and may conveniently take the form of powder or liquidsprays or inhalants, lozenges, throat paints, etc. For medication of theeyes or ears, the preparations may be presented as individual capsules,in liquid or semi-solid form, or may be used as drops, etc. Topicalapplications may be formulated in hydrophobic or hydrophilic bases asointments, creams, lotions, paints, powders, etc.

[0114] For veterinary medicine, the composition may, for example, beformulated as an intra-mammary preparation in either long acting orquick-release bases.

[0115] Useful dosages of the compounds of the invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949.

[0116] Generally, the concentration of the compound(s) of the inventionin a liquid composition, such as a lotion, will be from about 0.1-25wt-%, preferably from about 0.5-10 wt-%. The concentration in asemi-solid or solid composition such as a gel or a powder will be about0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

[0117] The compositions per unit dosage, whether liquid or solid maycontain from 0.1% to 99% of active material (compound I or saltsthereof), the preferred range being from about 10-60%. The compositionwill generally contain from about 15 mg to about 1,500 mg by weight ofactive ingredient based upon the total weight of the composition;however, in general, it is preferable to employ a dosage amount in therange of from about 250 mg to 1,000 mg. In parenteral administration theunit dosage is usually the pure compound in a slightly acidified sterilewater solution or in the form of a soluble powder intended for solution.Single dosages for injection, infusion or ingestion may be administered,i.e., 1-3 times daily, to yield levels of about 0.5-50 mg/kg, foradults.

[0118] The ability of a compound of the invention to function as atransferase inhibitor or blocker can be demonstrated using the testmethods described below, or using other tests which are well known inthe art. Representative compounds of formula I can be readily evaluatedas inhibitors the above mentioned transferases by, for example, relativeIC₅₀ analysis.

[0119] The present invention provides chemical syntheses of fluorescentanalogs of farnesol (FOH), farnesyl pyrophosphate (FPP), geranylgeraniol(GGOH), and geranylgeranyl pyrophosphate (GGPP). These analogs can bedesigned to display minimal differences from the natural substrates tomaximize the possibility that they can be incorporated into variouslipoproteins.

[0120] The present invention suggests in vitro testing of the compoundsof the present invention with prenyl transferases and their proteinsubstrates to demonstrate inhibition of modified proteins. For example,experiments with isolated Ras proteins, normal isoprenoid substrates,and farnesyl protein transferase should give unprenylated proteins.

[0121] The present invention suggests in vitro testing of the compoundsof the present invention with prenyl transferases and their proteinsubstrates to demonstrate formation of unnaturally modified proteins.For example, experiments with isolated Ras proteins and farnesyl proteintransferase should give “farnesylated” proteins carrying fluorescentlabels and provide protein standards for in vivo experiments.

[0122] The present invention suggests in vivo testing of the compoundsof the present invention in cell lines to determine if the fluorescentisoprenoid analogs serve as inhibitors for the enzymes that convertproteins to prenylated lipoproteins, ultimately resulting in theaccumulation of unmodified proteins and reduction in prenylated proteinsin living cells. These experiments can be accomplished in normal culturemedia or with cells depleted of natural isoprenoids (e.g., by treatmentwith lovastatin).

[0123] The present invention suggests in vivo testing of the compoundsof the present invention in cell lines to determine if the fluorescentisoprenoid analogs serve as substrates for the enzymes that convertproteins to prenylated lipoproteins, ultimately providing fluorescentprenylated proteins in living cells. These experiments can be done innormal culture media or with cells depleted of natural isoprenoids(e.g., by treatment with lovastatin).

[0124] The present invention provides chemical synthesis of analogs ofthe natural isoprenoids doubly modified to re-direct post-translationalprocessing and label the resulting proteins with fluorescent tags.

[0125] The present invention provides a method for the analysis ofcellular traffic in prenylated proteins, for example, using confocalmicroscopy to monitor localization of prenylated proteins carryingfluorescent labels. These experiments can be done initially underconditions as natural as possible and then in the presence ofrepresentative FPTase inhibitors or alternative substrates designed toredirect post-translational processing.

[0126] The following discussion, figures, and examples further describeand exemplify making and using the present invention.

[0127] Three prenylation motifs are known and each is recognized by aspecific prenyl transferase. A first prenyl transferase is the enzymefarnesyl protein transferase (FTase or FPTase) recognizes a carboxylterminal amino acid sequence described as a “—CAAX box,” where C iscysteine, A is any aliphatic amino acid, and X is serine, methionine,glutamine, or alanine. FPTase transfers a farnesyl group from farnesylpyrophosphate (FPP) to the sulfhydryl group of the cysteine. Theresultant protein is further processed by proteolytic cleavage of thethree C-terminal amino acids in a reaction catalyzed by the proteaseRCE1, and then methylation of the newly freed carboxyl group throughreaction catalyzed by the enzyme Icmt. The net effect of thesetransformations is transformation of a hydrophilic protein foundprimarily in the cytosol to a hydrophobic protein found primarily inassociation with a lipid membrane.

[0128] A second prenyl transferase (GGTase or GGPTase I) is closelyrelated to FPTase in its structure and substrate specificity. Thisenzyme transfers a geranylgeranyl group from geranylgeranylpyrophosphate (GGPP) to a cysteine sulfhydryl group in proteins bearinga —CAAX box where the terminal amino acid is a leucine. It is highlyselective for transfer of a geranylgeranyl group but also will bind FPPand catalyze transfer of a farnesyl group to some substrates.

[0129] A third enzyme known to transfer prenyl groups to proteins isgeranylgeranyl transferase II (GGTase II or GGPTase II) also referred toas Rab geranylgeranyl transferase. Until very recently this enzyme wasthought to have an absolute specificity for GGPP and not bind FPP, butthis has now been questioned. Its protein substrates differsignificantly from those of GGPTase I in that two cysteine residues arerequired at, or adjacent to, the carboxyl terminus in amino acidsequences such as —XXCC, —XCXC, or —CCXX. Finally, short peptidesbearing these sequences are not substrates for this enzyme, while shortsequences with the —CAAX box do serve as substrates for FPTase and/orGGPTase I.

[0130]FIG. 2 shows schematically the overall mechanism of RASfarnesylation involves FPTase catalyzed nucleophilic attack of the —SHgroup of the cysteine in the —CAAX box on the C-1 position of farnesylpyrophosphate. Whether by an S_(N) ¹ or S_(N) ² mechanism, or someintermediate variant possible only in the enzyme's active site, thisresults in displacement of pyrophosphate and formation of a new covalentbond between RAS and the farnesyl group. A inhibition strategy forinterrupting this process is one using compound analogs where aphosphate group is not readily lost. For example, variousfarnesylphosphonates, wherein a carbon-phosphorus bond joins thephosphoryl group to the farnesyl chain, incorporate this essentialproperty along with a highly desirable similarity to the naturalsubstrate.

[0131]FIG. 3 shows schematically an example of a reaction with certainother analog compounds which afford an unnaturally modified Ras proteinand which analog compound products or adducts may be unable to undergoretro-conjugate addition and therefore have greater stability andutility for inhibiting transferase enzymes and for probing the mechanismof various transferase enzymes and processes.

[0132] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1

[0133] PREPARATION OF FLUORESCENT ANALOG COMPOUNDS

[0134] As exemplified in the reaction scheme below, Fischeresterification of commercial anthranilic acid provides the correspondingamino ester 54. Reductive amination of amine 54 with aldehyde 55,derived from SeO₂ oxidation of geranyl acetate and subsequent MnO₂oxidation, gave the desired amine 56 and subsequent cleavage of theacetate gave the famesol analog 57. The resulting farnesol analog (57)has a much greater degree of metabolic stability compared to otherpreviously prepared analogs since this compound employs an amine linkageto bind an anthranilic acid to the terpenoid chain.

[0135] Compound 57 is highly fluorescent and it readily penetrates cellmembranes to afford fluorescent cells. Compound 58 can be used in enzymeexperiments with FPTase and Ras in order to obtain standard Ras proteinslabeled with the fluorescent isoprenoid analog. Similarly compound 62can be used with GGPTase I or II and proteins such as Rho B to obtainother protein standards. Protein standards obtained from in vitroexperiments can then be used to analyze cell lysates for the formationof analogous proteins through in vivo experiments. Compound 58 ¹HNMR(D₂O) δ 7.6 (1H), 7.2 (1H), 6.4 (2H), 5.4 (1H), 5.2 (1H), 4.4 (2H),3.6 (3H), 3.4 (2H), 2.0 (2H), 1.8 (2H), 1.6 (3H), 1.4 (3H); and ³¹P NMR−6.9 (d, 1P) −9.75 (d, 1P).

EXAMPLE 2

[0136] PREPARATION OF FLUORESCENT ANALOG COMPOUNDS

[0137] A second family of isoprenoid analogs can be prepared as shownbelow and have useful fluorescent properties based onaminonaphthalenesulfonic acids related to the dansyl group. Commercial5-aminonaphthalene sulfonic acid (63) is first protected as its Bocderivative 64, and then converted to the N,N-dimethylsulfonamide (66)via the corresponding sulfonyl chloride (65). After cleavage of the Bocgroup, reductive amination is conducted with amine 67 and the aldehyde(68), derived from oxidation of prenyl acetate, affords the famesolanalog 69. The corresponding alcohol (70) can be used directly in wholecell experiments, and converted to the corresponding pyrophosphate (71)for either in vitro or in vivo use. The geranylgeraniol analog 73, aswell as its pyrophosphate 74, can be obtained through parallel reactionsif the geranyl acetate derivative 55 is used in the reductive amination,for example, to afford amine 72. Compound (73) ¹H NMR(CDCl₃) δ 8.20 (dd,1H, J=6.23, 1.16 Hz), 8.12 (d, 1H, J=7.68 Hz), 8.09 (d, 1H, J=7.85 Hz),7.49 (dd, 1H, J=7.30, 0.93 Hz), 7.46 (dd, 1H, J=8.80, 1.53 Hz), 6.68 (d,1H, J=7.71 Hz), 5.48 (dt, 1H, J=5.81, 1.1 Hz), 5.38 (dt, 1H, J=68, 1.11Hz), 4.15 (d, 1H, J=7.05 Hz), 4.10 (d, 1H, J=7.55 Hz), 3.82 (s, 2H),2.82 (s, 6H), 2.24 (dd, 2H, J=14.48, 7.11 Hz), 2.09 (t, 2H, 7.78 Hz),1.76 (s, 3H), 1.68 (s, 3H); ¹³C NMR(CDCl₃) δ 144.1, 139.1, 133.1, 131.8,130.3, 130.1, 128.9, 126.3, 126.0, 124.4, 123.8, 122.5, 114.1, 105.8,59.3, 51.9, 39.1, 37.4(2C), 26.0, 16.2, 14.9; and High Resolution MassSpectra m/z obsd. 403.2059 (M+H)⁺, calcd for C₂₂H₃₁N₂O₃S 403.2055.

[0138] Advantages of naphthalene-based analogs include the dansyl-likecompounds which can provide prenylated proteins with fluorescentproperties that differ from those of the anthranilate-based compounds.One of ordinary skill in the art would expect an absorption maximum atabout 340 nm but an emission maximum of about 540 nm for dansylderivatives. In addition, if the dansyl-based compounds can serve assubstrates, it might be possible to construct farnesol analogs with oneset of spectral properties (e.g., based on anthranilate derivatives) andgeranylgeraniol derivatives with a second set of properties (e.g., basedon the larger dansyl group). The availability of fluorescent derivativesof both isoprenoid series as metabolic probes enables one to establishthe ratio of farnesylation to geranylgeranylation under differentconditions.

EXAMPLE 3

[0139] The following illustrate representative pharmaceutical dosageforms, containing a compound of formula I (‘Compound X’), fortherapeutic or prophylactic use in humans. (i) Tablet 1 mg/tablet‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0 Croscarmellose sodium 12.0Microcrystalline cellulose 92.5 Magnesium stearate 3.0 300.0 (ii) Tablet2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose 410.0 Starch50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0 500.0 (iii)Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0(iv) Injection 1 (1 mg/ml) mg/ml ‘Compound X’ (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0 N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL (v) Injection 2 (10 mg/ml)mg/ml ‘Compound X’ (free acid form) 10.0 Monobasic sodium phosphate 0.3Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.0 01 N Sodiumhydroxide solution q.s. (pH adjustment to 7.0-7.5) Water for injectionq.s. ad 1 mL (vi) Aerosol mg/can ‘Compound X’ 20.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

[0140] The above formulations may be obtained by conventional procedureswell known in the pharmaceutical art. Although specific quantities of“Compound X” are shown in the above illustrative examples, it is to beunderstood that the compounds can be present in any ratio provided thefinal formulation possesses the desired formulation properties.

[0141] All publications, patents, and patent documents are incorporatedby reference herein in their entirety. The invention has been describedwith reference to various specific and preferred embodiments andtechniques. However, it should be understood that many variations andmodifications may be made while remaining within the spirit and scope ofthe invention.

REFERENCES

[0142] Inhibition of Farnesyltransferase with A-176120, a Novel andPotent Farnesyl Pyrophosphate Analogue. Tahir, S. K.; Gu, W. Z.; Zhang,H. C.; Leal, J.; Lee, J. Y.; Kover, P.; Saeed, B.; Cherian, S. P.;Devine, E.; Cohen, J.; Warner, R.; Wang, Y. C.; Stout, D.; Arendsen, D.L.; Rosenberg, S.; Ng, S.C. Eur. J. Cancer, 2000, 36, 1161-1170.

[0143] Farnesyl Diphosphate-Based Inhibitors of RAS Farnesyl-ProteinTransferase. Patel, D. V.; Schmidt, R. J.; Biller, S. A.; Gordon, E. M.;Robinson, S. S.; Manne, V. J. Med. Chem. 1995, 38, 2906-2921.

[0144] Cocrystal Structure of Protein Farnesyltransferase Complexed WithA Farnesyl Diphosphate Substrate. Long, S. B.; Casey, P. J.; Beese, L.S. Biochemistry, 1998, 37, 9612-9618.

[0145] On the Stereochemical Course of Human Protein-FarnesylTransferase. Mu, Y. -Q.; Omer, C. A.; Gibbs, R. A. J. Am. Chem. Soc.1996, 118, 1817-1823.

[0146] Synthesis And Conformational Analysis of DI-C-13-Labeled FarnesylDiphosphate Analogs. Zahn, T. J.; Ksebati, M. B.; Gibbs, R. A.Tetrahedron Lett. 1998, 39, 3991-3994. Evaluation of isoprenoidconformation in solution and in the active site of protein-farnesyltransferase using carbon-13 labeling in conjunction with solution- andsolid-state NMR. Zahn T. J.; Eilers, M.; Guo, Z. M.; Ksebati, M. B.;Simon, M.; Scholten, J. D.; Smith, S. O.; Gibbs, R. A. J. Am. Chem. Soc.2000, 122, 7153-7164.

[0147] Preparation of (2E,6E)-10,11-Dihydrofarnesol Via A(Bisphenyl)Dithioacetal Reduction. Mechelke, M. F.; Wiemer, D. F.Tetrahedron Lett. 1998, 39, 9609-9612.

[0148] Stereochemical Analysis of the Reaction Catalyzed By YeastProtein Farnesyltransferase. Edelstein, R. L.; Weller, V. A.; Distefano,M. D.; Tung, J. S. J. Org. Chem. 1998, 63, 5298-5299.

[0149] Synthesis of Farnesol Analogues Through Cu(I)-MediatedDisplacements of Allylic THP Ethers By Grignard Reagents. Mechelke, M.F.; Wiemer, D. F. J. Org. Chem. 1999, 64, 4821-4829.

[0150] Novel Phosphonylphosphinyl (P—C—P—C—) Analogs of BiochemicallyInteresting Diphosphates—Synthesis And Properties of P—C—P—C— Analogs OfIsopentenyl Diphosphate And Dimethylallyl Diphosphate. McClard, R. W.;Fujita, T. S.; Stremler, K. E.; Poulter, C. D. J. Am. Chem. Soc. 1987,109, 5544-5545.

[0151] Steady-State Kinetic Mechanism of RAS Farnesyl-ProteinTransferase. Pompliano, D. L.; Rands, E.; Schaber, M. D.; Mosser, S. D.;Anthony, N. J.; Gibbs, J. B. Biochemistry 1992, 31, 3800-3807.

[0152] A PD(0)-Catalyzed Route To 13-Methylidenefarnesyl Diphosphate.Gibbs, R. A.; Krishnan, U. Tetrahedron Lett. 1994, 35, 2509-2512.

[0153] Cuprate-Mediated Synthesis And Biological Evaluation ofCyclopropyl- and Tert-Butylfarnesyl Diphosphate Analogs. Mu, Y.; Gibbs,R. A.; Eubanks, L. M.; Poulter, C. D. J. Org. Chem. 1996, 61, 8010-8015.

[0154] Farnesyl-Derived Inhibitors of RAS Farnesyl Transferase. Kang, M.S.; Stemerick, D. M.; Zwolshen, J. H.; Harry, B. S.; Sunkara, P. S.;Harrison, B. L. Biochem. Biophys. Res. Comm. 1995, 217, 245-249. f)Synthesis of Pyrophosphonic Acid Analogs of Farnesyl Pyrophosphate.Valentijn, A. R. P. M.; van den Berg, O.; van der Marel, G. A.; Cohen,L. H.; van Boom, J. H. Tetrahedron, 1995, 51, 2099-2108.

[0155] Photoactive Analogs of Farnesyl Pyrophosphate ContainingBenzoylbenzoate Esters: Synthesis and Application to PhotoaffinityLabeling of Yeast Protein Farnesyltransferase. Gaon, I.; Turek, T. C.;Weller, V. A.; Edelstein, R. L.; Singh, S. K.; Distefano, M. D. J. Org.Chem. 1996, 61, 7738-7745.

[0156] Stereochemistry-Dependent Inhibition of RAS Farnesylation ByFarnesyl Phosphonic Acids. Hohl, R. J.; Lewis, K. A.; Cermak, D. M.;Wiemer, D. F. Lipids, 1998, 33, 39-46.

[0157] Phosphonate and Bisphosphonate Analogs of Farnesyl PyrophosphateAs Potential Inhibitors of Farnesyl Protein Transferase. Holstein, S.A.; Cermak, D. M.; Wiemer, D. F.; Lewis, K.; Hohl, R. J. Bioorganic &Medicinal Chemistry 1998, 6, 687-694.

[0158] Synthesis of Nonracemic DimethylAlpha-(Hydroxyfarnesyl)Phosphonates Via Oxidation of DimethylFarnesylphosphonate With (Camphorsulfonyl)Oxaziridines. Cermak, D. M.;Du, Y.; Wiemer, D. F. J. Org. Chem. 1999, 64, 388-393.

[0159] Novel famesol and geranylgeraniol analogues: A potential newclass of anticancer agents directed against protein prenylation. Gibbs,B. S.; Zahn, T. J.; Mu, Y. Q.; Sebolt-Leopold, J. S.; Gibbs, R. A. J.Med. Chem. 1999, 42, 3800-3808.

[0160] Design and synthesis of a transferable farnesyl pyrophosphateanalogue to Ras by protein farnesyltransferase. Chehade, K. A. H.;Andres, D. A.; Morimoto, H.; Spielmann, H. P. J. Org. Chem. 2000, 65,388-393.

[0161] Phase I and Pharmacokinetic Study of Farnesyl Protein TransferaseInhibitor R115777 in Advanced Cancer. Zujewski, J.; Horak, I. D.; Bol,C. J.; Woestenborghs, R.; Bowden, C.; End, D. W.; Piotrovsky, V. K.;Chiao, J.; Belly, R. T.; Todd, A.; Kopp, W. C.; Kohler, D. R.; Chow, C.;Noone, M.; Hakim, F. T.; Larkin, G.; Gress, R. E.; Nussenblatt, R. B.;Kremer, A. B.; Cowan, K. H. J. Clin. Oncol.2000, 18, 927-941.

[0162] Clinical and Biological Activity of the FarnesyltransferaseInhibitor R115777 in Adults with Refractory and Relapsed AcuteLeukemias: A Phase I Clinical-Laboratory Correlative Trial. Karp, J. E.;Lancet, J. E.; Kaufmann, S. H.; End, D. W.; Wright, J. J.; Bol, K.;Horak, I.; Tidwell, M. L.; Leisveld, J.; Kottke, T. J.; Ange, D.;Buddharaju, L.; Gojo, I.; Highsmith, W. E.; Belly, R. T.; Hohl, R. J.;Rybak, M. E.; Thibault, A.; and Rosenblatt, J. Blood. 2001, 97,3361-3369.

[0163] A Phase I Trial of the Farnesyl Transferase Inhibitor SCH66336:Evidence for Biological and Clinical Activity. Adjei, A. A.; Erlichman,C.; Davis, J. N.; Cutler, D. L.; Sloan, J. A.; Marks, R. S.; Hanson, L.J.; Svingen, P. A.; Atherton, P.; Bishop, W. R.; Kirschmeier, P.;Kaufman, S. H. Cancer Res.2000, 60, 1871-1877.

[0164] Discovery and structure-activity relationships ofimidazole-containing tetrahydrobenzodiazepine inhibitors offarnesyltransferase. Ding, C. Z.; Batorsky, R.; Bhide, R.; Chao, H. G.J.; Cho, Y.; Chong, S.; Gullo-Brown, J.; Guo, P.; Kim, S. H.; Lee, F.;Leftheris, K.; Miller, A.; Mitt, T.; Patel, M.; Penhallow, B. A.; Ricca,C.; Rose, W. C.; Schmidt, R.; Slusarchyk, W. A.; Vite, G.; Yan, N.;Manne, V.; Hunt, J. T. J. Med. Chem. 1999, 42, 5241-5253.

[0165] Discovery of(R)-7-cyano-2,3,4,5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulfonyl)-1H-1,4-benzodiazepine(BMS-214662), a farnesyltransferase inhibitor with potent preclinicalantitumor activity. Hunt, J. T. et al. J. Med. Chem. 2000, 43,3587-3595.

[0166] Discovery of a Series of Cyclohexylethylamine-Containing ProteinFarnesyltransferase Inhibitors Exhibiting Potent Cellular Activity.Henry, K. J; Wasicak, J.; Tasker, A. S.; Cohen, J.; Ewing, P.; Mitten,M.; Larsen, J. J.; Klavin, D. M.; Swenson, R.; Ng, S. C.; Saeed, B.;Cherian, S.; Sham, H.; Rosenberg, S. J. Med. Chem. 1999, 42, 4844-4852.

[0167] A Peptidomimetic Inhibitor of Farnesyl:Protein Transferase Blocksthe Anchorage-Dependent and -Independent Growth of Human Tumor CellLines. Sepp-Lorenzino, L.; Ma, Z.; Rands, E.; Kohl, N. E.; Gibbs, J. B.,Oliff, A.; Rosen, N. Cancer Research, 1995, 55, 5302-5309.

[0168] Geranylgeranylated RhoB Mediates Suppression of Human Tumor CellGrowth By Farnesyltransferase Inhibitors. Du, W.; Prendergast, G. C.Cancer Res. 1999, 59, 5492-5496.

[0169] Both Farnesylated and Geranylgeranylated RhoB Inhibit MalignantTransformation and Suppress Human Tumor Growth in Nude Mice. Chen, Z.;Sun, J.; Pradines, A.; Favre, G.; Adnane, J.; Sebti, S. M. J. Biol.Chem. 2000, 275, 17974-17978.

[0170] RhoB Alteration is Necessary for Apoptotic and AntineoplasticResponses to Farnesyltransferase Inhibitors. Liu, A.; Du, W.; Liu, J.P.; Jessell, T. M.; Prendergast, G. C. Mol. Cell. Biol. 2000, 20,6105-6113.

[0171] Farnesyl transferase inhibitors block the farnesylation of CENP-Eand CENP-F and alter the association of CENP-E with the microtubules.Ashar, H. R.; James, L.; Gray, K.; Carr, D.; Black, S.; Armstrong, L.;Bishop, W. R.; Kirschmeier, P. J. Biol. Chem. 2000, 275, 30451-30457.

[0172] Farnesyl Transferase Inhibitors Cause Enhanced MitoticSensitivity to Taxol and Epothilones. Moasser, M. M.; Sepp-Lorenzino,L.; Kohl, N. E.; Oliff, A.; Balog, A.; Su, D. S.; Danishefsky, S. J.;Rosen, N. Proc. Natl. Acad.Sci. USA, 1998, 20, 139-148.

[0173] Antitumor Efficacy of a Novel Class of Nonthiol-containingPeptidomimetic Inhibitors of Farnesyltransferase andGeranylgeranyltrasferase I: Combination Therapy with the CytotoxicAgents Cisplatin, Taxol, and Gemcitabine. Sun, J.; Blaskovich, M. A.;Knowles, D.; Qian, Y.; Ohkanada, J.; Bailey, R. D.; Hamilton, A. D.;Sebti, S. M. Cancer Res. 1999, 59, 4919-4926.

[0174] The Phosphoinositide 3-OH Kinase/AKT2 Pathway as a CriticalTarget for Farnesyltransferase-Induced Apoptosis. Jiang, K.; Coppola,D.; Crespo, N. C.; Nicosia, S. V.; Hamilton, A. D.; Sebti, S. M.; Cheng,J. Q. Mol. Cell. Biol. 2000, 20, 139-148.

[0175] Cdk Inhibitors, Roscovitine and Olomoucine, Synergize withFarnesyltrasferase Inhibitor (FTI) to Induce Efficient Apoptosis ofHuman Cancer Cell Lines. Edamatsu, H.; Gau, C. L.; Nemoto, T.; Guo, L.;Tamanoi, F. Oncogene, 2000, 19, 3059-3068.

[0176] Inhibition of Protein Geranylgeranylation Causes a Superinductionof Nitric-oxide Synthase-2 by Interleukin-1β in Vascular Smooth MuscleCells. Finder, J. D.; Litz, J. L.; Blaskovich, M. A.; McGuire, T. F.;Qian, Y.; Hamilton, A. D.; Davies, P.; Sebti, S. M. J. Biol. Chem. 1997,272, 13484-13488.

[0177] Farnesyl Pyrophosphate Synthase is the Molecular Target ofNitrogen-Containing Bisphosphonates. VanBeek, E.; Pieterman, E.; Cohen,L.; Lowik, C.; Papapoulous, S. Biochem. Biophys. Res. Commun. 1999, 264,108-111.

[0178] HMG CoA Reductase Inhibitor-Induced Myotoxicity: Pravastatin andLovastatin Inhibit the Geranylgeranylation of Low-Molecular WeightProteins in Neonatal Rat Muscle Cell Culture. Flint, O. P.; Masters, B.A.; Gregg, R. E.; Durham, S. K. Toxicol. Appl. Pharmacol. 1997, 145,99-110.

[0179] Nitrogen-Containing Bisphosphonates Inhibit the MevalonatePathway and Prevent Post-Translational Prenylation of GTP-BindingProteins, Inducing Ras. Luckman, S. P.; Hughes, D. E.; Coxon, F. P.;Russell, R. G. G.; Rogers, M. J. J. Bone Miner. Res. 1998, 13, 581-589.

[0180] Preparation of diterpenoid derivatives as inhibitors ofprenyl-protein transferase. Anthony, N. J.; Gomez, R. P.; Omer, U.S.Pat. No. 5,574,025, 1996. (CA 125: 86936.)

[0181] Intramolecular Fluorescence Enhancement: A Continuous Assay ofRAS Farnesyl: Protein Transferase. Pompliano, D. L.; Gomez, R. P.;Anthony, N. J. J. Am. Chem. Soc. 1992, 114, 7945-7946.

[0182] 2-(Acyloxy)ethylphosphonate Analogues of Prenyl Pyrophosphates:Synthesis and Biological Characterization. Cermak, D. M.; Wiemer, D. F.;Lewis, K.; Hohl, R. J. Bioorg. Med. Chem. 2000, 8, 2729-2737.

[0183] Lewis, K., Du, Y., Yang, L., Wiemer, D. F. Hohl, R. J. Effect ofCombinations of Lovastatin and Specific Protein IsoprenylationInhibitors on RAS and Mitogen Activated Protein (MAP) Kinase Activities.In Preparation.

[0184] Chemo-enzymatic synthesis of fluorescent Rab 7 proteins: Tools tostudy vesicular trafficking in cells. Owen D J, Goody R S, et al.,Angew. Chem., Int. Ed. Eng. 1999, 38, 509-512.

[0185] Phosphorylation of Isoprenoid Alcohols. Jo Davisson, V.;Woodside, A. B.; Neal, T. R.; Stremler, K. E.; Muehlbacher, M.; Poulter,C. D. J. Org. Chem. 1986, 51, 4768-4779.

[0186] Synthesis and DNA-Binding Properties of a Cisplatin AnalogContaining a Tethered Dansyl Goup. Hartwig, J. F.; Pil, P. M.; Lippard,S. J. J. Am. Chem. Soc. 1992, 114, 8292-8293, and references citedtherein.

[0187] The Dansyl Group as a Molecular Probe for the HistochemicalLocalization of a Synthetic Fibronectin-Related Peptide. Yamamoto, Y.;Katow, H.; Sofuku, S. Chemistry Letters 1994, 8, 1379-1382.

[0188] Enzymatic modification of proteins with a geranylgeranylisorenoid. Casey P. J.; Thissen, J. A.; Moomaw, J. F. Proc. Natl. Acad.Sci. U.S.A. 1991, 88, 8631-8635.

[0189] The dually acylated NH2-terminal domain of gi 1 alpha issufficient to target a green flourescent protein reporter to thecaveolin-enriched plasma membrane domians. Palmitoylation of caveolin-1is required for the recognition to dually acylated g-protein alphasubunits in vivo. Galbiati, F.; Volonte, D.; Meani, D.; Milligan, G.;Lublin, D. M.; Lisanti, M. P.; Parenti, M. J. Biol. Chem. 1999, 274,5843-5850.

[0190] Regulation of insulin stimulated GLUT4 translocation by Munc 18cin 3T3L1 adipocytes. Thurmond, D. C.; Ceresa, B. P.; Okada, S.;Elmendorf, J. S.; Coker, K.; Pessin, J. E. J. Biol. Chem. 1998, 273,33876-33883.

[0191] Inhibition of hydroxymethylglutaryl coenzyme A reductase activityinduces a paradoxical increase in DNA synthesis in myeloid leukemiacells. Hohl, R. J.; Larson, R. A.; Mannickarottu, V.; Yachnin, S. Blood1991, 77, 1064-1070.

[0192] Stereoelectronic Effects in Biomolecules. Gorenstein, D. G. Chem.Rev. 1987, 87, 1047-1077. b) Stereoelectronic Effects on theConformation and Kinetics of Nucleophilic Displacement-Reactions inEpimeric 6-Membered Ring Phosphonate Diesters. Chang, J. W. A.;Gorenstein, D. G. Tetrahedron, 1987, 43, 5187-5196.

[0193] Targeted inactivation of the isoprenylcysteinecarboxymethyltransferase gene causes mislocation of K-Ras in mammaliancells. Bergo, M. O.; Leung, G. K.; Ambroziack, P.; Otto, J. C.; Casey,P. J.; Young, S. G. J. Biol. Chem. 2000, 275, 17605-17610.

[0194] Mutational and Biochemical Analysis of Plasma Membrane TargetingMediated by the Farnesylated, Polybasic Carboxy Terminus of K-ras4B.Roy, M. O.; Leventis, R.; Silvius, J. R. Biochemistry 2000, 39,8298-8307.

What is claimed is:
 1. A compound of formula I:

wherein: X is independently O or S; R₁ is a detectable group; R₂ isindependently OH, (C₁-C₁₀)alkanoyloxy, —O—P(═O)(—OR_(a))₂,—O—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂, —CH₂—O—P(═O)(—OR_(a))₂,—CH₂—O—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂, —CH₂—P(═O)(—OR_(a))₂,—CH{—P(═O)(—OR_(a))₂}₂, —CH₂—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂,—CH═CH{—P(═O)(—OR_(a))₂}, or —CH═C{—P(═O)(—OR_(a))₂}₂; each R_(a) isindependently hydrogen, (C₁-C₁₀)alkyl, (C₁-C₁₀)alkanoyl,(C₁-C₁₀)alkanoyloxy, (C₁-C₁₀)alkoxycarbonyl, or —CH₂—O—(C₁-C₁₀)alkanoyl;n is independently 1, 2, or 3; or a pharmaceutically acceptable saltthereof.
 2. The compound of claim 1 wherein the detectable group is arylor Het, optionally substituted with one or more substituentsindependently selected from —COOR_(b), —S(O)_(n)NR_(b)R_(b), halo,cyano, nitro, aryl, heterocycle, (C₁-C₁₀)alkoxy, (C₂-C₆)alkenyl,—C(═O)NR_(b)R_(b), —OC(═O)NR_(b)R_(b), —NR_(b)R_(b), or —S(O)_(n)R_(b),where each R_(b) is independently hydrogen, (C₁-C₁₀)alkyl, or(C₁-C₁₀)alkanoyl.
 3. The compound of claim 2 wherein aryl or Het isphenyl, indenyl, naphthyl, anthracenyl, or anthranil, which aryl or Hetis optionally substituted with one or more substituents independentlyselected from —COOR_(b), —S(O)_(n)NR_(b)R_(b), halo, cyano, nitro, aryl,heterocycle, (C₂-C₆)alkenyl, —C(═O)NR_(b)R_(b), —OC(═O)NR_(b)R_(b),—NR_(b)R_(b), or —S(O)_(n)R_(b), where each R_(b) is independentlyhydrogen, (C₁-C₁₀)alkyl, or (C₁-C₁₀)alkanoyl.
 4. The compound of claim 1wherein R₁ is substituted phenyl.
 5. The compound of claim 1 wherein R₁is phenyl substituted with —COOR_(b).
 6. The compound of claim 1 whereinR₁ is 2-methoxycarboxy phenyl.
 7. The compound of claim 1 wherein R₁ issubstituted naphthyl.
 8. The compound of claim 1 wherein R₁ is naphthylsubstituted with a —S(O)_(n)NR_(b)R_(b).
 9. The compound of claim 1wherein R₁ is naphthyl substituted at the 5-position with a—S(O)_(n)NR_(b)R_(b) substituent.
 10. The compound of claim 1 wherein R₁is 5-N,N′—dimethylaminosulfonyl naphthy-1-yl.
 11. The compound of claim1 wherein R₂ is OH.
 12. The compound of claim 1 wherein R₂ is(C₁-C₁₀)alkanoyloxy.
 13. The compound of claim 1 wherein R₂ is—O—P(═O)(—OR_(a))₂.
 14. The compound of claim 1 wherein R₂ is—O—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂.
 15. The compound of claim 1 whereinR₂ is —CH₂—O—P(═O)(—OR_(a))₂.
 16. The compound of claim 1 wherein R₂ is—CH₂—O—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂.
 17. The compound of claim 1wherein R₂ is —CH₂—P(═O)(—OR_(a))₂.
 18. The compound of claim 1 whereinR₂ is —CH{—P(═O)(—OR_(a))₂}₂.
 19. The compound of claim 1 wherein R₂ is—CH₂—P(═O)(—OR_(a))—O—P(═O)(—OR_(a))₂.
 20. The compound of claim 1wherein R₂ is —CH═CH{—P(═O)(—OR_(a))₂}.
 21. The compound of claim 1wherein R₂ is —CH═C {—P(═O)(—OR_(a))₂}₂.
 22. The compound of claim 1wherein R_(a) is hydrogen.
 23. The compound of claim 1 wherein R_(a) is—C(═O)—CH₃.
 24. The compound of claim 1 wherein R_(a) is —CH₃.
 25. Thecompound of claim 1 wherein R_(a) is —CH₂—O—(C₁-C₆)alkanoyl.
 26. Thecompound of claim 2 wherein R_(b) is hydrogen.
 27. The compound of claim2 wherein R_(b) is —CH₃.
 28. The compound of claim 1 wherein n is
 1. 29.The compound of claim 1 wherein n is
 2. 30. The compound of claim 1wherein n is
 3. 31. The compound of claim 1 wherein X is —O—.
 32. Thecompound of claim 1 wherein X is —S—.
 33. A pharmaceutical compositioncomprising a compound as described in claim 1 and a pharmaceuticallyacceptable diluent or carrier.
 34. A method of treating cancer,comprising administering to a mammal afflicted with cancer, an amount ofa compound as described in claim 1 effective to treat said cancer.
 35. Amethod of inhibiting a prenylation transferase enzyme or synthase enzymecomprising contacting the enzyme with an effective amount of a compoundas described in claim
 1. 36. A method of accessing the metabolic statusof an enzyme comprising: contacting the enzyme with an effective amountof a mixture of a farnesol analog compound and a geraniol orgeranylgeraniol analog compound as described in claim 1; and measuringthe relative ratio of farnesylation to geranylgeranylation of thefamesol and the geraniol or geranylgeraniol analog compoundsaccomplished by the enzyme.
 37. A compound as described in claim 1 foruse in medical therapy or diagnosis.
 38. The compound of claim 37wherein the therapy or diagnosis is treating cancer.
 39. The use of acompound as described in claim 1 for the manufacture of a medicamentuseful for the treatment of cancer.
 40. The use of a compound asdescribed in claim 1 for the manufacture of a medicament useful forinhibiting prenylation transferase enzymes in a mammal.
 41. A proteinconjugate comprising a protein linked to a fluorescent fragment of acompound of claim 1.